The human pathogen, Neisseria gonorrhoeae (Ngo) is capable of utilizing human proteins as sources of necessary metals such as iron (Fe) and zinc (Zn). The ability to employ human transferrin (hTf) as an Fe source requires expression of a TonB-dependent, integral outer membrane transporter (TbpA), and a surface- exposed lipoprotein (TbpB). Homologous but distinct TonB-dependent transporters are produced by Ngo that enable the pathogen to utilize other innate immunity proteins as sole Zn sources. Like the proteins that enable use of hTf (TbpA and TbpB), the other four under-characterized TonB-dependent transporters (TdTs) are well- conserved among Neisseria species, are not subject to high-frequency variation and are not produced by the human host or other non-Neisseria commensal bacteria. The current proposal is focused on exploring the vaccine potential of the TdTs with the ultimate goal being a multi-component vaccine that targets multiple essential nutrient acquisition systems. The overarching hypothesis is that a rationally-designed vaccine consisting of engineered TdTs combined with experimentally-selected adjuvants will provide immunologic cross-protection against colonization and disease caused by diverse Ngo strains. The specific aims are as follows: Aim 1. Structural, bioinformatics and mutagenic analysis of under-characterized TdTs. We intend to characterize the structures of four TdTs with and without ligand to guide mutagenesis and vaccine efforts. We will assess the presence and variability of these TdTs among diverse Ngo strains. And we will mutagenize the TdTs to eliminate ligand-binding functions. Aim 2. Optimizing adjuvants and delivery modes for elicitation of specific immune responses. Adjuvants, delivery routes, doses and schedules will be tested for optimal anti- TdT responses in mice. Immunological responses will be defined by broadly testing humoral and cellular immunity factors. Aim 3. Test for protective efficacy of TdT-based vaccines in new humanized infection models. Optimized antigens, adjuvants, routes and schedules will be tested for protection in humanized mouse models of lower female genital tract infection, pelvic inflammatory disease, male urethritis and nasopharyngeal infection. Immunological correlates and determinants of protection in these new animal models of infection will be characterized. Aim 4. Evaluate rationally designed vaccines for cross-protection in all models of infection. Optimized, rationally-designed vaccine formulations will be tested for protection against a broad group of Ngo strains, including antimicrobial resistant ?superbug? strains. Immunological correlates of protection will be validated with these strains in all of the humanized mouse models of infection. These studies are significant since they may lead to the development of an efficacious vaccine against a recalcitrant pathogen that has developed resistance to existing therapeutic methods. These studies are innovative because they will employ structure-guided vaccine design to develop ligand-binding incompetent vaccine antigens, which will be tested for protection in novel humanized mouse models of infection.